Aptamer sensors with continuous solute passivation

ABSTRACT

A continuous sensing device for measuring at least one analyte included in a sample fluid is provided. The device 1000a includes a plurality of aptamers located in a sensor fluid 18, at least one electrode 1050, and at least one element configured to passivate the at least one electrode by continuous solute passivation. The device may further include at least one cleaning element 1092 to clean the at least one electrode.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/082,834, filed on Sep. 24, 2020; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/082,999, filed on Sep. 24, 2020; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/083,029, filed on Sep. 24, 2020; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/085,484, filed on Sep. 30, 2020; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/122,071, filed on Dec. 7, 2020; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/122,076, filed on Dec. 7, 2020; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/136,262, filed on Jan. 12, 2021; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/150,667, filed on Feb. 18, 2021; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/150,677, filed on Feb. 18, 2021; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/150,712, filed on Feb. 18, 2021; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/150,856, filed on Feb. 18, 2021; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/150,865, filed on Feb. 18, 2021; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/150,894, filed on Feb. 18, 2021; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/150,944, filed on Feb. 18, 2021; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/150,953, filed on Feb. 18, 2021; claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/150,986, filed on Feb. 18, 2021; and claims the benefit of the filing date of U.S. Patent Application Ser. No. 63/197,674, filed on Jun. 7, 2021, the disclosures of each of which are incorporated by reference herein in their entireties.

BACKGROUND OF THE INVENTION

This section is intended to introduce the reader to various aspects of art that may be related to various aspects of the present invention, which are described and/or claimed below. This discussion is believed to be helpful in providing the reader with background information to facilitate a better understanding of various aspects of the present invention. Accordingly, it should be understood that these statements are to be read in this light, and not as admissions of prior art.

Electrochemical aptamer sensors can identify the presence and/or concentration of an analyte of interest via the use of an aptamer sequence that specifically binds to the analyte of interest. These sensors include aptamers attached to an electrode, wherein each of the aptamers has a redox active molecule (redox couple) attached thereto. The redox couple can transfer electrical charge to or from the electrode. When an analyte binds to the aptamer, the aptamer changes shape, bringing the redox couple closer to or further from, on average, the electrode. This results in a measurable change in electrical current that can be translated to a measure of concentration of the analyte.

Major unresolved challenges for electrochemical aptamer sensors include the relatively short lifetime of the sensors and drift of the sensors (i.e., a general decrease in the accuracy of the sensors over time). These issues are especially pronounced for applications where continuous operation is required (“continuous operation” referring to multiple measurements taken over time by the same device). One of the challenges presented is the presence of species in a sample fluid other than those attributable to the target analyte that would otherwise generate the same sensor signal generated by detection of target analyte. This creates a background signal that inhibits the ability to detect small concentration analytes, and can also create inaccurate readings.

To attempt to resolve these issues, many electrochemical aptamer sensor devices include a blocking layer to reduce background electrochemical current at the working electrode. However, to date, these blocking layers have not been effective enough in reducing the background current that confounds detection of low concentrations of analyte and creates background electrochemical current which can damage the electrode or aptamers or blocking layers over time. Background current for aptamer sensors tested in whole blood, serum, interstitial fluid, or other protein and solute rich fluids is less than background current in buffer solution or dilute biofluids, because they contain species such as albumin which can non-specifically bond to the blocking layer or electrode surface and thereby help further passivate the electrode. However, these same biofluids contain proteases, enzymes, and other solutes that can also degrade the aptamer sensor over time. Fouling of sensor electrodes used in analyte detection is a significant problem such that the lifetime of sensor electrodes is significantly shortened. Devices and methods that overcome the issues of sensor degradation, inaccuracy, fouling, and short lifetime are needed.

SUMMARY OF THE INVENTION

Certain exemplary aspects of the invention are set forth below. It should be understood that these aspects are presented merely to provide the reader with a brief summary of certain forms the invention might take and that these aspects are not intended to limit the scope of the invention. Indeed, the invention may encompass a variety of aspects that may not be explicitly set forth below.

Many of the drawbacks and limitations stated above can be resolved by creating novel and advanced interplays of chemicals, materials, sensors, electronics, microfluidics, algorithms, computing, software, systems, and other features or designs, in a manner that affordably, effectively, conveniently, intelligently, or reliably brings sensing technology into proximity with sample fluids containing at least one analyte of interest to be measured.

Various aspects of the disclosed invention are directed to aptamer sensors (including those capable of continuous operation) that leverage the benefit of solute passivation while preventing the negative effects of direct exposure to biofluids or sample fluids.

A continuous sensing device for measuring at least one analyte included in a sample fluid is provided. The device includes a plurality of aptamers located in a sensor fluid, at least one electrode, and at least one element configured to passivate the at least one electrode by continuous solute passivation.

A continuous sensing device for measuring at least one analyte included in a sample fluid is provided. The device includes a plurality of aptamers located in a sensor fluid. The device further includes at least one electrode. The device further includes a redox tag coupled to the aptamer, the redox tag configured to change an electron transfer to between the electrode and the aptamer in response to the analyte coupling to the aptamer. The device further includes at least one cleaning element configured to clean a surface of the electrode.

A method of sensing at least one analyte included in a sample fluid is also provided. The method includes bringing an analyte included in the sample fluid into contact with an aptamer included in a sensor fluid, the contact of the aptamer with the analyte resulting in a change in the electron transfer between a redox tag and an electrode. The method further includes measuring the change in electron transfer between the redox tag and the electrode. The method further includes passivating the electrode by continuous solute passivation with at least one element.

BRIEF DESCRIPTION OF THE DRAWINGS

The objects and advantages of the disclosed invention will be further appreciated in light of the following detailed descriptions and drawings in which:

FIG. 1A is a cross-sectional view of a device in accordance with principles of the disclosed invention.

FIG. 1B is a cross-sectional view of an alternate embodiment of a device in accordance with principles of the disclosed invention.

FIG. 2A is a schematic showing a prior art portion of an aptamer sensor device having a passivating layer and an aptamer attached to an electrode.

FIG. 2B is a schematic showing the aptamer and passivating layer portions of the aptamer sensor device of FIG. 2A degrading over time.

FIG. 3 is a schematic of yet another embodiment of a device in accordance with principles of the present invention.

FIG. 4 is a graph showing the effect of a membrane in an aptamer sensor device on percentage of solute retention versus molecular weight of the solute.

FIG. 5A is a schematic of yet another embodiment of a device in accordance with principles of the present invention.

FIG. 5B is a schematic showing an alternate embodiment of an aptamer with attached redox tag that can be used with devices in accordance with principles of the disclosed invention.

FIG. 5C is a schematic showing yet another alternate embodiment of an aptamer with attached redox tag that can be used with devices in accordance with principles of the disclosed invention.

FIG. 5D is a schematic showing yet another alternate embodiment of an aptamer with attached redox tag that can be used with devices in accordance with principles of the disclosed invention.

FIG. 5E is a schematic showing yet another alternate embodiment of an aptamer with attached redox tag that can be used with devices in accordance with principles of the disclosed invention.

FIG. 6A is a schematic of yet another embodiment of a device in accordance with principles of the present invention.

FIG. 6B is a schematic showing an alternate embodiment of an aptamer with attached redox tag that can be used with devices in accordance with principles of the disclosed invention.

FIG. 6C is a schematic showing yet another alternate embodiment of an aptamer with attached redox tag that can be used with devices in accordance with principles of the disclosed invention.

FIG. 7 is a schematic of yet another embodiment of a device in accordance with principles of the present invention.

FIG. 8 is a schematic of yet another embodiment of a device in accordance with principles of the present invention.

FIG. 9 is a schematic of yet another embodiment of a device in accordance with principles of the present invention.

FIG. 10A is a schematic of one embodiment of a device in accordance with principles of the present invention wherein electrodes may be mechanically cleaned in situ.

FIG. 10B is a schematic of another embodiment of a device in accordance with principles of the present invention wherein electrodes may be mechanically cleaned in situ.

FIG. 11A is a graph showing raw chronoamperometric scans of current versus time for cortisol in an exemplary device.

FIG. 11B is a graph showing normalized current gain for three sensors versus concentration of cortisol.

DEFINITIONS

As used herein, “continuous sensing” with a “continuous sensor” means a sensor that changes in response to changing concentration of at least one solute in a solution such as an analyte. Similarly, as used herein, “continuous monitoring” means the capability of a device to provide multiple measurements of an analyte over time.

As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, pH, size, concentration or percentage is meant to encompass variations of ±20% in some embodiments, ±10% in some embodiments, ±5% in some embodiments, ±1% in some embodiments, ±0.5% in some embodiments, and ±0.1% in some embodiments from the specified amount, as such variations are appropriate to perform the disclosed method.

As used herein, the term “electrode” means any material that is electrically conductive such as gold, platinum, nickel, silicon, conductive liquid infused materials such as ionic liquids, PEDOT:PSS, conductive oxides, carbon, boron-doped diamond, nanotubes or nanowire meshes, or other suitable electrically conducting materials.

As used herein, the term “blocking layer” or “passivating layer” means a homogeneous or heterogeneous layer of molecules on an electrode which alter the electrochemical behavior of the electrode. Examples include a monolayer of mercaptohexanol on a gold electrode or as another example natural small-molecule solutes in blood that form a layer on a carbon electrode. A passivating layer as taught herein may also be referred to as a fouling layer if the solutes are endogenous and may also be referred to as a “layer of endogenous solutes.”

As used herein, the term “solute passivation” means passivation of an electrode based on aptamers based on exogenous or endogenous molecules in a sample fluid or a sensor fluid. Exogenous molecules, for example, may include mercaptohexanol on a gold or platinum electrode, while endogenous molecules may include for example peptides, hormones, amino-acids, or other solutes found in a sample fluid such as blood.

As used herein, the term “continuous solute passivation” means solute passivation of an electrode where the endogenous or exogenous molecules may degrade over time or move back into solution and leave the electrode, which opens up the electrode for additional solutes to passivate such that a passivating layer is maintained continuously on the electrode.

As used herein, the term “aptamer” means a molecule that undergoes a conformation or binding change as an analyte binds to the molecule, and which satisfies the general operating principles of the sensing method as described herein. Such molecules are, e.g., natural or modified DNA, RNA, or XNA oligonucleotide sequences, spiegelmers, peptide aptamers, and affimers. Modifications may include substituting unnatural nucleic acid bases for natural bases within the aptamer sequence, replacing natural sequences with unnatural sequences, or other suitable modifications that improve sensor function but which behave analogous to traditional aptamers. Two or more aptamers bound together can also be referred to as an aptamer (i.e. not separated in solution). Aptamers can have molecular weights of at least 1 kDa, 10 kDa, or 100 kDa.

As used herein, the term “redox tag” or “redox molecule” means any species such as small or large molecules with a redox active portion that when brought adjacent to an electrode can reversibly transfer at least one electron with the electrode. Redox tag or molecule examples include methylene blue, ferrocene, quinones, or other suitable species that satisfy the definition of a redox tag or molecule. In some cases a redox tag or molecule is referred to as a redox mediator. Redox tags or molecules may also exchange electrons with other redox tags or molecules.

As used herein, the term “optical tag” or “fluorescent tag” means any species that fluoresces in response to an optical source such as LED and whose fluorescence is detectable by a photodetector such as a photodiode. Example fluorescent tags include fluorescein and may be used in combination with other fluorescent tags or optical quenchers such a black-hole quencher dyes to change the fluorescence of the optical tag.

As used herein, the term “signaling aptamer” means an aptamer that is tagged with a redox active molecule or tag and/or contains a redox active portion itself and which provides a change in electrochemical signal when it is released from an anchor aptamer.

As used herein, the term “anchor aptamer” means an aptamer that that can bind to a signaling aptamer, and when bound to the signaling aptamer changes at least one property of the bound vs. unbound signaling aptamer such as molecular weight, diffusion coefficient, charge state, being floating in solution vs. being immobilized, or some other property which achieves the stated effect for the signaling aptamer.

As used herein, the term “folded aptamer” means an aptamer that along its length associates with itself in one or more locations creating a three-dimensional structure for the aptamer that is distinct from an “unfolded aptamer” that is a freely floating and oscillating strand of aptamer. Aptamers can also be partially folded or partially unfolded in structure or in time spent in the folded vs. unfolded states. Multiple folding configurations are also possible.

As used herein, the term “analyte” means any solute in a solution or fluid which can be measured using a sensor. Analytes can be small molecules, proteins, peptides, electrolytes, acids, bases, antibodies, molecules with small molecules bound to them, DNA, RNA, drugs, chemicals, pollutants, or other solutes in a solution or fluid.

As used herein, the term “membrane” means a polymer film, plug of hydrogel, liquid-infused film, tiny pore, or other suitable material which is permiselective to transport of a solute through the membrane by solute parameters such as size, charge state, hydrophobicity, physical structure, or other solute parameters than can enable permiselectivity. For example, a dialysis membrane is permselective by passing small solutes but not large solutes such as proteins. Membranes as understood herein need not be multiporous, for example a nanotube or nanopore can act as a permiselective filter and is therefore considered part of a membrane as understood for the present invention.

As used herein, the term “sample fluid” means any solution or fluid that contains at least one analyte to be measured.

As used herein, the term “sensor fluid” means a solution or fluid that differs from a sample solution by at least one property, and through which the sensor solution and the sample solution are therefore separated but are in fluidic connection through at least one pathway such as a membrane. The sensor solution comprises at least one aptamer as a solute.

As used herein, the term “reservoir fluid” means a solution or fluid that differs from a sample solution by at least one property, and through which the sensor solution and the reservoir solution are in fluidic connection through at least one pathway such as a membrane or a pin-hole connection. A reservoir fluid may have multiple function in a device, for example, by introducing a solute continuously or as needed by diffusion equilibrium into the sensor fluid, or for example removing unwanted solutes from a sensor fluid and acting as a “waste removal element”.

As used herein, a “device” comprises at least one sensor based on at least one aptamer, at least one sensor solution, and at least one sample solution. Devices can sense multiple samples and be in multiple configurations such as a device to measure a pin-prick of blood, or a microneedle or in-dwelling sensor needle to measure interstitial fluid, or a device to measure saliva, tears, sweat, or urine sensor, or a device to measure water pollutants or food processing solutes, or other devices which measure at least one analyte found in a sample solution.

DETAILED DESCRIPTION OF THE INVENTION

One or more specific embodiments of the present invention will be described below. In an effort to provide a concise description of these embodiments, all features of an actual implementation may not be described in the specification. It should be appreciated that in the development of any such actual implementation, as in any engineering or design project, numerous implementation-specific decisions must be made to achieve the developers' specific goals, such as compliance with system-related and business-related constraints, which may vary from one implementation to another. Moreover, it should be appreciated that such a development effort might be complex and time consuming, but would nevertheless be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill having the benefit of this disclosure.

Certain embodiments of the disclosed invention show sensors as simple individual elements. It is understood that many sensors require two or more electrodes, reference electrodes, or additional supporting technology or features which are not captured in the description herein. Sensors are preferably electrical in nature, but may also include optical such as a LED or laser excitation source and a photodetector, chemical, mechanical, or other known biosensing mechanisms. Sensors can be in duplicate, triplicate, or more, to provide improved data and readings. Sensors may provide continuous or discrete data and/or readings. Certain embodiments of the disclosed invention show sub-components of what would be sensing devices with more sub-components needed for use of the device in various applications, which are known (e.g., a battery, antenna, adhesive), and for purposes of brevity and focus on inventive aspects, such components may not be explicitly shown in the diagrams or described in the embodiments of the disclosed invention. All ranges of parameters disclosed herein include the endpoints of the ranges.

The present invention cures many of the problems of the prior art discussed above. For example, as previously described, fouling of sensor electrodes used in analyte detection is a significant problem such that the lifetime of sensor electrodes is significantly shortened. Aspects of the present invention, however, provide a device including a cleaning element, which cleans the sensor electrode. As a result, the working lifetime of the sensor electrode is significantly increased because the cleaning element is able to remove waste or fouling species from the surface of the electrode. In some device embodiments of the present invention, at least one cleaning element is included such that the cleaning element is configured to clean a surface of the electrode of waste or fouling species.

With reference to FIGS. 1A and 1B, exemplary embodiments of devices in accordance with principles of the disclosed invention are shown. Referring first to FIG. 1A, a device 100 is shown as being placed partially in-vivo into the skin 12 of a subject. Skin 12 includes the epidermis 12 a, the dermis 12 b, and the subcutaneous or hypodermis 12 c. The device 100 includes a feature 112 that allows for access to sample fluids from the subject. Such sample fluids may include interstitial fluid (from the dermis 12 b) and/or blood (from a capillary 12 d). In the embodiment shown in FIG. 1A, the feature 112 includes a plurality of microneedles (which may be formed of metal, polymer, semiconductor, glass, or other suitable material). Each of the microneedles 112 projects from a first substrate 108. And each microneedle 112 may include a hollow lumen 132. The device 100 also includes a second substrate 110 (which may be a material such as polymer or glass) having an electrode 150 adjacent thereto. An optional passivating layer 120 may be adjacent to electrode 150, such that electrode 150 is positioned between passivating layer 120 and second substrate 110. Passivating layer 120 includes a compound such as mercaptohexanol or may comprise natural solutes that have diffused into the device 100 from the dermis 12 b.

As can be seen in FIG. 1A, a defined volume 130 is present between first substrate 108 and passivating layer 120. It will be recognized by those of ordinary skill in the art that defined volume 130 does not necessarily have to be defined by first substrate 108 and passivating layer 120—and in embodiments where passivating layer is absent, volume 130 may be defined by first substrate 108 and electrode 150; or, alternatively, may be defined by first substrate 108 and second substrate 110. A sensor fluid 18 may be present within volume 130 (as shown in FIG. 1A). Further, as can be seen in the embodiment of FIG. 1A, at least one membrane 136 is present between first substrate 108 and passivating layer 120, and is positioned adjacent first substrate 108. The at least one membrane 136 may be of various materials or substances—such as a dialysis membrane or hydrogel, for example. In the particular embodiment shown in FIG. 1A, portions of the membrane 136 overlie the boundary between volume 130 and lumens 132 of each microneedle 112. Due to this positioning of membrane 136, volume 130 includes sensor fluid 18, and lumens 132 include sample fluid 14—such as interstitial fluid from dermis 12 b or blood from capillary 12 d. Together the total volume provided by volume 130 and lumens 132 can be a microfluidic component such as channels, a hydrogel, or other suitable material. A diffusion or other fluidic pathway exists from the sample fluid 14, such as interstitial fluid or blood, into volumes 132, 130.

Another embodiment of a device 100 is shown in FIG. 1B. This embodiment also includes first and second substrates 108, 110, microneedles 112 having lumens 132, electrode 150, passivating layer 120, defined volume 130 and at least one membrane 136. As can be seen from FIG. 1B, in this embodiment, electrode 150 and passivating layer 120 are recessed in second substrate 110 (as opposed to the configuration shown in FIG. 1A). Thus, volume 130 is defined by first substrate 108 and combination of second substrate 110 and passivating layer 120. Further, the embodiment as shown in FIG. 1B includes a plurality of membranes 136, with each membrane 136 positioned in a distal end of each lumen 132 of each microneedle 112. Due to this positioning of membranes 136, both volume 130 and lumens 132 include a sensor fluid 18 (and sample fluid is present in, and obtainable from, dermis 12 b and capillary 12 d, for example).

Alternative arrangements and materials to those discussed above with respect to FIGS. 1A and 1B are possible, such as using a single needle or hydrogel polymer microneedles. In addition, one or more of the features of device 100 or the entire device 100 could be implanted into the body and perform similarly as described herein. Furthermore, a device 100 could be fully outside the body, if for example sampling a fluid such as sweat or tears.

Turning now to FIGS. 2A and 2B, where like numerals refer to like features, a portion of a prior art device 200 is shown. Referring to FIG. 2A, an aptamer sensor includes a passivating or blocking layer 248 (including a compound such as mercaptohexanol) attached to an electrode 250 (made from a material such as gold), and having at least one aptamer 270 that is attached to the electrode 250, such as by being thiol-bonded to the electrode 250. The aptamer 270 has at least one redox tag or molecule 240, such as methylene blue, associated therewith. The device 200 is shown as being positioned in a sample fluid 14, such as blood or interstitial fluid (for example). This prior art device 200 may have an analyte (not shown) that binds with the aptamer 270, thereby changing the availability of the redox tag 240 to the electrode 250, such as by bringing it closer to, or further from, the electrode 250. Conventional aptamer sensors can be limited in performance because an aptamer that is bound to an electrode often has a weaker binding affinity to an analyte than an aptamer that is free in solution. In addition, as shown in FIG. 2B, the sensors can degrade as the aptamer 270 and/or blocking layer 248 degrades over time (e.g., chemical degradation, or detaching from the electrode 250). Also, because such prior art devices 200 have relied on exogenous molecules (e.g., mercaptohexanol) for passivation, the passivation layer 248 can also become thicker with fouling from solutes (such as albumin) in the sample fluid 14.

Thus, and with reference now to FIG. 3 , where like numerals refer to like features, an embodiment of the disclosed invention that improves on the prior art devices and reduces or eliminates drawbacks with such devices is shown. To that end, FIG. 3 shows a device 300 (or at least a portion thereof) that includes an electrode 350 and at least one membrane 336 which separates a sample fluid 14 from a sensor fluid 18. The sensor fluid 18 contains a plurality of aptamers 370 having redox tags 340. The electrode 350 may include a passivating layer 348. The passivating layer 348 may comprise one or more endogeneous solutes 16 from the sample fluid 14 itself (or, as initially prepared, the passivating layer 348 may be prepared from molecules that are known to be endogenous to the sample fluid to be tested). Examples of such endogenous molecules 16 include small molecules such as amino acids, hormones, metabolites, or peptides. (Thus, the device 300 shown in FIG. 3 differs from that described above in prior art FIGS. 2A and 2B in that the prior art device described above included an aptamer and an exogenous molecule, such as mercaptohexanol. Similarly, electrode 350 could also contain a passivation layer 348 comprised, at least in part, by an exogenous molecule such as hexanethiol or mercaptohexanol. But, even in that case, the passivation layer 348 could detach from the electrode 350 and be in need of replacement.) By including endogeneous molecules 16 in the passivation layer 348, longer lifetime of the device 300 is achieved because endogenous molecules 16 can leave the electrode 350 as shown by arrow 392 and another endogenous molecule 16 can replace that now-missing molecule as shown by arrow 390. Thus, in a sense, the very molecules in the sample fluid 14 can be used to “repair” the passivation layer as it degrades, thereby extending the life of the device. (As mentioned above, these endogenous molecules can originate from the sample fluid itself, be already present as a deliberate component of the sensor fluid, or could be a mix of the two.) As a non-limiting example, membrane 336 is able to pass in small solutes (e.g., <1 kDa)—for example, an analyte such as cortisol—and passivating solutes 16, such as amino-acids and peptides, but retains the aptamer 370 (with redox tag 340) which is often >10 kDa in molecular weight. If the aptamer 370 with redox tag 340 were not retained by the membrane 336, then aptamer 370 with redox tag 340 could be lost into the body and no longer able be able to provide a measurement of the analyte.

Further, although solutes (endogenous molecules 16) allow for endogenous passivation, they can also cause the passivation layer 348 to continue to grow in thickness over time, which at some point will decrease electron transfer from the redox tags 340. This not only can reduce signal strength from the sensor, but give errors in reading of the concentration of analytes. Solutions to these challenges will be presented after discussion of FIGS. 1A-9 .

An example of the analysis of the use of a membrane to pass small solutes (small target analyte) while retaining aptamers within device is shown with reference to FIG. 4 , which shows an illustrative plot of solute retention for a membrane such as membrane 336. This is an example only, and shows that if measuring a small analyte such as cortisol (<400 Da) and using a large aptamer (>10 kDa or even >50 kDa) a membrane could be highly permeable to the analyte and poorly permeable to the aptamer. Thus, for example, in various embodiments, membranes of the present invention may have molecular weight cutoffs (i.e., the molecular weight above which a molecule will not easily pass through the membrane) that are at least one of <300 Da, <1000 Da, <3 kDa, <10 kDa, <30 kDa, <100 kDa, <300 kDa. Larger molecular weight cut-off membranes will require larger sized aptamers to prevent the aptamers from potentially escaping the device.

Several additional embodiments will be discussed below. In these additional embodiments, an increase in availability of the redox tag to the electrode can occur as a result of aptamer binding analyte, or, alternatively, without aptamer binding to an analyte. And even though each of the embodiments discussed below (and their respective figures) may show one specific example, the other the embodiments of the invention are not so limited (e.g., the various aptamer/redox tag types can be used across the various embodiments of devices disclosed herein, and vice versa.

Turning now to FIGS. 5A-5E, where like numerals refer to like features: FIG. 5A shows a portion of a device 500 including a substrate 510, a sensor fluid 18, a plurality of aptamers 570 with redox tags 540 free in the sensor fluid 18, a passivation layer 548 of endogenous solutes 16, and an electrode adjacent the substrate 510. Though not part of the device, analytes 19 are also depicted as present in the sensor fluid of the device. The schematic shown in FIG. 5A also depicts an electron transfer event that occurs between a redox tag 540 and the electrode 550. This is shown generally at reference numeral 598, and is a non-limiting example depicting that electron transfer 598 from a redox tag 540 occurs in an increased amount, or frequency, or rate, when aptamer 570 binding to analyte 19 occurs (e.g., as shown in the figure, when analyte is not bound to aptamer, the redox tag is not available—or is less available—to the electrode, due to, for example, a conformation of the aptamer that hinders or prevents such transfer when not bound to analyte; conversely, when aptamer binds analyte, the conformation of aptamer may change in a manner that positions the redox tag for electron transfer). In various embodiments, aptamer binding to analyte can provide changes in electron transfer and redox current (compared to baseline transfer and current—i.e., transfer/current in the absence of analyte binding) of greater than 5%, greater than 10%, greater than 20%, greater than 50%, greater than 100%, or greater than 200%. For the embodiments illustrated herein, non-limiting examples of electrical measurement techniques may include voltammetry, square wave voltammetry, amperometry, chronoamperometry, coulometry, chronocoulometry, with a preferred embodiment being square wave voltammetry.

FIG. 5B schematically depicts another example of an aptamer 570 with attached redox tag 540 (that differs from the aptamer 570/redox tag 540 schematically shown in FIG. 5A). The embodiment of the aptamer 570 in FIG. 5B is designed such that the redox tag 540 is more available for electron transfer with the electrode 550 in the absence of any analyte 19 binding to the aptamer 570 (high electron transfer—or high ET). Conversely, when analyte 19 binds to the aptamer 570 of FIG. 5B, the redox tag 540 is less available for electron transfer with the electrode 550 (e.g., the redox tag 540 is less exposed—low ET).

FIG. 5C schematically depicts yet another example of an aptamer with attached redox tag 540 (that differs from the aptamer 570/redox tag 540 schematically shown in FIGS. 5A and 5B). The embodiment shown in FIG. 5C is designed with two aptamer portions: a signaling aptamer 572 and an anchor aptamer 574. A redox tag 540 is associated with (such as by being attached to) the signaling aptamer 572. The anchor aptamer 574 includes a portion that has affinity for, and thus can bind, analyte 19. When analyte 19 is not bound to the anchor aptamer 574 (left side of FIG. 5C), the signaling aptamer 572 remains associated with the anchor aptamer 574, and so the redox tag 540 on signaling aptamer 572 is less available for electron transfer with the electrode 550 (low ET). However, once the anchor aptamer 574 binds to analyte 19 (right side of FIG. 5C), signaling aptamer 572 is released from anchor aptamer 574, and the redox tag 540 becomes more available for electron transfer with the electrode 550 (high ET). It will be recognized that the device of the embodiment of FIG. 5C has a plurality of aptamers—and thus includes a plurality of signaling aptamers 572, and a plurality of anchor aptamers 574. As described above, each anchor aptamer of the plurality of anchor aptamers is adapted to bind to analyte. In one embodiment, each signaling aptamer of a majority of the plurality of signaling aptamers is bound to a respective anchor aptamer when a majority of anchor aptamers are not bound to any analyte. (This may occur, for example, prior to the introduction of any analyte.) Once analyte is introduced (such as when a sample fluid is introduced into the device—e.g., by being introduced into the sensor fluid of the device), at least a subset of anchor aptamers from the plurality of anchor aptamers then binds to analyte. When this occurs, a subset of signaling aptamers (from the total plurality of aptamers) dissociates from the anchor aptamers—and the redox tag becomes more available for electron transfer with the electrode.

Further, while the embodiment shown in FIG. 5C depicts analyte 19 binding to anchor aptamer 574 and redox tag 540 on signaling aptamer 572, in an alternate embodiment analyte binding may occur with signaling aptamer (signaling aptamer having redox tag), and binding of analyte to signaling aptamer may serve to release signaling aptamer from anchor aptamer (such as by change in conformation of signaling aptamer). Even further configurations as possible, as understood by those skilled in the art of aptamers. To maximize the signal gain (change in signal) signaling aptamer 572 concentration will typically be less than or equal to the anchor aptamer 574 concentration else the signaling aptamer can cause increased background signal with or without the presence of analyte.

FIG. 5D schematically depicts yet another example of an aptamer with attached redox tag 540 (that differs from the aptamers/redox tags schematically shown in FIGS. 5A, 5B, and 5C). The embodiment of the aptamer 570 in FIG. 5D has both a redox tag 544 and a redox quencher 542 associated therewith (such as by being bound to the aptamer 570). When analyte 19 is not bound to the aptamer 570, the redox tag 544 and redox quencher 542 are spatially separated (left side of FIG. 5D) thereby allowing for greater electron transfer between redox tag 544 and electrode 550 (high ET). However, once the aptamer 570 binds to analyte 19 (right side of FIG. 5D), the redox tag 544 and redox quencher 542 are brought into closer proximity with one another, thereby causing less electron transfer between redox tag 544 and electrode 550 (low ET). Numerous quenchers are possible, including anthraquinone-based redox molecules that can be self-quenching when two of such identical molecules are brought close together (monomer vs. dimer).

FIG. 5E schematically depicts yet another example of an aptamer with attached redox tag 540 (that differs from the aptamers/redox tags schematically shown in FIGS. 5A, 5B, 5C, and 5D). The embodiment of the aptamer 570 in FIG. 5E has both a first redox tag 546 and a second redox tag 548 associated therewith (such as by being bound to the aptamer 570). When analyte 19 is not bound to the aptamer 570, the first and second redox tags 546, 548 are spatially separated (left side of FIG. 5E) thereby allowing for greater electron transfer between first and second redox tags 546, 548 and electrode 550 (high ET). However, once the aptamer 570 binds to analyte 19 (right side of FIG. 5E), the first and second redox tags 546, 548 are brought closer together and the electron transfer from one of the redox tags 546, 548 to the electrode 550 is altered due to a two-step mediated electron transfer process, or other effect, for two redox tags brought into close proximity. These changes in electron transfer are depicted in the voltammograms as shown as 546 a and 548 a. A non-limiting example of redox tags that enable the embodiment of FIG. 5E include methylene blue and ferricyanide.

Turning now to FIGS. 6A-6C, where like numerals refer to like features: FIG. 6A shows a portion of a device 600 that includes a substrate 610, at least first and second electrodes 650, 652, a passivation layer 648 including endogenous solutes 16, a sensor fluid 18 (which, in the embodiment illustrated in FIG. 6A is inside an optional hydrogel 638), a plurality of aptamers 670 having redox tags 640 (free in solution), and a diffusion or iontophoretic pathway 694. Though not part of the device, analytes 19 are also depicted as present in the sensor fluid of the device. FIG. 6A also schematically depicts electron transfer that can occur between redox tags 640 and the first and second electrodes 650, 652. As can be seen in FIG. 6A, as a non-limiting example, electron transfer 698 from the redox tags 640 in an increased amount, or frequency, or rate, when analyte 19 is bound to the aptamer 670. For example, when analyte is bound to aptamer, the hydrodynamic radius or size of the aptamer is smaller and therefore providing a faster diffusion coefficient, which results in the redox tag being more available for electron transfer with the electrodes (such a version will be discussed in greater detail below with respect to FIG. 6B); or, for example, when analyte is not bound to aptamer, the redox tag is not available—or is less available—to the electrode, due to, for example, a conformation of the aptamer that hinders or prevents such transfer when not bound to analyte; conversely, when aptamer binds analyte, the conformation of aptamer may change in a manner that positions the redox tag for electron transfer. In various embodiments, aptamer binding to analyte can provide changes in electron transfer and redox current (compared to baseline transfer and current—i.e., transfer/current in the absence of analyte binding) of greater than 5%, greater than 10%, greater than 20%, greater than 50%, greater than 100%, or greater than 200%. For the embodiments illustrated in FIGS. 6A-6C, non-limiting examples of electrical measurement techniques may include voltammetry, square wave voltammetry, amperometry, chronoamperometry, coulometry, chronocoulometry, with a preferred embodiment being amperometry.

FIG. 6B schematically depicts another example of an aptamer 670 with attached redox tag 640 (that differs from the aptamer 670/redox tag 640 schematically shown in FIG. 6A). The embodiment of the aptamer in FIG. 6B is designed such that the redox tag 640 is less available for electron transfer with the electrodes 650, 652 in the absence of analyte binding to aptamer (left side of FIG. 6B), because of a longer diffusion time between the first and second electrodes 650, 652 where the analyte can undergo redox recycling (e.g., one electrode is a reducing electrode, one electrode is an oxidizing electrode). However, when analyte 19 binds to the aptamer 670 (right side of FIG. 6B), the hydrodynamic radius or size of the aptamer is smaller and therefore providing a faster diffusion coefficient, and therefore redox tag 640 is more available for electron transfer with the first and second electrodes 650, 652. The binding of analyte 19 transforms the aptamer 670 between a long unfolded aptamer 670 (in the absence of analyte 19 binding) and an aptamer 670 with three stems when analyte 19 binds to aptamer 670.

As described above, with respect to FIG. 6A, a non-limiting example of an environment within a device 600 may include an optional hydrogel. In such an embodiment, the hydrogel 638 (such as agar or polyacrylamide) is added to further distinguish diffusion times between aptamers 670 bound to analyte 19 and aptamers 670 not bound to analyte. This is because the hydrogel 638 creates a more tortuous and size-selective diffusion pathway than a pure fluid would by itself. For example, an aptamer 670 that fully dissociates could be modified to have a significant change in hydrodynamic radius (R), which changes its diffusion coefficient (D) according to D=kT/(6 π η R). This equation is for diffusion in pure solution; a dense hydrogel 638 can be added to further distinguish the diffusion of the unfolded aptamer vs. the folded aptamer. The resulting current between the redox recycling electrodes is proportional as I∝D C/z, where C is the concentration of the aptamer 670 and z the electrode-to-electrode distance. With respect to changes in signal gain, the diffusion length of oligonucleotides (aptamers) varies with length to the ˜0.6^(th) power, and a 15 kDa protein that is globular/unfolded can have a change in R of 2.15/3.65.

FIG. 6C schematically depicts yet another example of an aptamer 670 with attached redox tag 640 (that differs from the aptamer 670/redox tag 640 schematically shown in FIGS. 6A and 6B). The embodiment of the aptamer in FIG. 6C is designed with two aptamer portions: a signaling aptamer 672 and an anchor aptamer 674. A redox tag 640 is associated with (such as by being attached to) the signaling aptamer 672. The anchor aptamer 674 includes a portion that has affinity for, and thus can bind, analyte 19. When analyte 19 is not bound to the anchor aptamer 674 (left side of FIG. 6C), the signaling aptamer 672 remains associated with the anchor aptamer 674, and so the redox tag 640 on signaling aptamer 672 is less available for electron transfer with the first and second electrode 650, 652 (as the combined signaling and anchor aptamers 672, 674 will exhibit slower diffusion in sensor solution and hydrogel). However, once the anchor aptamer 674 binds to analyte 19 (right side of FIG. 6C), signaling aptamer 672 is released from anchor aptamer 674, and the redox tag 640 becomes more available for electron transfer with the first and second electrodes 650, 652 (as the liberated signaling aptamer 672 will exhibit more rapid diffusion in sensor solution and hydrogel). Further, while the embodiment shown in FIG. 6C depicts analyte 19 binding to anchor aptamer 674 and redox tag 640 on signaling aptamer 672, in an alternate embodiment analyte binding may occur with signaling aptamer (signaling aptamer having redox tag), and binding of analyte to signaling aptamer may serve to release signaling aptamer from anchor aptamer (such as by change in conformation of signaling aptamer).

It will be recognized that when the device shown in FIG. 6A uses the embodiment of aptamers of FIG. 6C, it will include a plurality of signaling aptamers 672, and a plurality of anchor aptamers 674. As described above, each anchor aptamer of the plurality of anchor aptamers is adapted to bind to analyte. In one embodiment, each signaling aptamer of a majority of the plurality of signaling aptamers is bound to a respective anchor aptamer when a majority of anchor aptamers are not bound to any analyte. (This may occur, for example, prior to the introduction of any analyte.) Once analyte is introduced (such as when a sample fluid is introduced into the device—e.g., by being introduced into the sensor fluid of the device), at least a subset of anchor aptamers from the plurality of anchor aptamers then binds to analyte. When this occurs, a subset of signaling aptamers (from the total plurality of aptamers) dissociates from the anchor aptamers—and the redox tag becomes more available for electron transfer with the electrode.

With further reference to FIG. 6C, in addition to changes in diffusion coefficient, the larger the effective sphere for the aptamer the less likely it will experience electron transfer with an electrode (with a first principles estimation based on the inverse of sphere area, proportional to 1/R{circumflex over ( )}2). This example is simply to show that two factors can be at play for embodiments of the present invention, both distance of the redox tag to the electrode and diffusion time to/from the electrode. This diffusion time to an electrode applies other embodiments as well, where for example with a chronoamperometric response for an aptamer the total current baseline could remain higher or reach baseline more quickly as diffusion coefficient for the aptamers increases. This diffusion time to an electrode may also impact interrogation methods such as square wave voltammetry, as aptamer that is near the electrode can contribute to the signal as well if it is able to diffuse to the electrode during each square window (during each voltage pulse that is applied). The first and second electrodes 650 and 652 can be closely spaced via interdigitation or other suitable technique, and, in such an embodiment, may be within less than 50 μm, less than 10 μm, less than 2 μm, or less than 0.4 μm distant of each other.

With reference to FIG. 7 where like numerals refer to like features, another embodiment in accordance with aspects of the present invention is shown. As can be seen in FIG. 7 , a portion of a device 700 is shown, and includes a substrate 710, at least one electrode 750, a passivation layer 748 including endogenous solutes 16, a sensor fluid 18, a plurality of aptamers having redox tags 740 (free in the sensor solution), and a poorly-mobile or non-mobile material 738 in the sensor fluid 18. Though not part of the device, analytes 19 are also depicted as present in the sensor fluid of the device.

The aptamers/redox tags component of the embodiment of FIG. 7 is similar to that shown in FIGS. 5C and 6C, and includes two aptamer portions: a signaling aptamer 772 and an anchor aptamer 774. A redox tag 740 is associated with (such as by being attached to) the signaling aptamer 772. The anchor aptamer 774 includes a portion that has affinity for, and thus can bind, analyte 19. As can be seen in FIG. 7 , the anchor aptamer 774 is immobilized via linkage 739 to the poorly or non-mobile material 738. The poorly-mobile or non-mobile material 738 may comprise various materials, such as a hydrogel. In one non-limiting example, the material 738 could be a hydrogel such as polyacrylamide and the linker be a molecule such as acrydite that is attached to the anchor aptamer at a terminal end or other location. In an alternate embodiment, the anchor aptamer could be cross-linked with other anchor aptamers or the anchor aptamer made so large (e.g., >100 kDa) such that it is effectively immobile in a dense hydrogel 738.

Still referring to FIG. 7 , when analyte 19 is not bound to the anchor aptamer 774, the signaling aptamer 772 remains associated with the anchor aptamer 774, and so the redox tag 740 on signaling aptamer 772 is less available for electron transfer with the electrode 750 (because the combined signaling and anchor aptamers 772, 774 will be poorly-mobile or non-mobile in the sensor fluid due to anchor aptamer 774 being linked to material 738). However, once the anchor aptamer 774 binds to analyte 19, the signaling aptamer 772 is released from anchor aptamer 774 (as indicated by arrow 796), and the redox tag 740 becomes more available for electron transfer with the electrode 750 (because the liberated signaling aptamer 772 will exhibit more rapid diffusion in sensor solution as it is no longer complexed with the anchor aptamer 774 that is linked to poorly-mobile or non-mobile material 738). Further, while the embodiment shown in FIG. 7 depicts analyte 19 binding to anchor aptamer 774 and redox tag 740 on signaling aptamer 772, in an alternate embodiment analyte binding may occur with signaling aptamer (signaling aptamer having redox tag), and binding of analyte to signaling aptamer may serve to release signaling aptamer from anchor aptamer (such as by change in conformation of signaling aptamer).

It will be recognized that the device of the embodiment of FIG. 7 has a plurality of aptamers—and thus includes a plurality of signaling aptamers 772, and a plurality of anchor aptamers 774. As described above, each anchor aptamer of the plurality of anchor aptamers is adapted to bind to analyte. In one embodiment, each signaling aptamer of a majority of the plurality of signaling aptamers is bound to a respective anchor aptamer when a majority of anchor aptamers are not bound to any analyte. (This may occur, for example, prior to the introduction of any analyte.) Once analyte is introduced (such as when a sample fluid is introduced into the device—e.g., by being introduced into the sensor fluid of the device), at least a subset of anchor aptamers from the plurality of anchor aptamers then binds to analyte. When this occurs, a subset of signaling aptamers (from the total plurality of aptamers) dissociates from the anchor aptamers—and the redox tag becomes more available for electron transfer with the electrode.

With reference to FIG. 8 , where like numerals refer to like features, another embodiment in accordance with aspects of the present invention is shown. As can be seen in FIG. 8 , a portion of a device 800 is shown, and includes a substrate 810, at least one electrode 850, a membrane 838, a sensor fluid 18, a plurality of aptamers having redox tags 740 (free in the sensor fluid). Though not part of the device, analytes 19 are also depicted as present in the sensor fluid of the device. The aptamers/redox tags component of the embodiment of FIG. 8 is similar to that shown in FIGS. 5C, 6C, and 7 , and includes two aptamer portions: a signaling aptamer 882 and an anchor aptamer 884. A redox tag 840 is associated with (such as by being attached to) the signaling aptamer 882. The anchor aptamer 884 includes a portion that has affinity for, and thus can bind, analyte 19. The membrane 838 exhibits selective permeability based on size, charge, or at least one solute property, and is able to pass a signaling aptamer 882 but not a signaling aptamer that is attached to a larger anchor aptamer 884. Thus, the membrane 838 impacts the availability of the redox couple 840 to the electrode 850. For example, a signaling aptamer could have a radius of 3 nm/2 nm in folded/unfolded states and an anchor aptamer have 27/7 nm in folded/unfolded state, creating a difference in size of ˜3-10× when a signaling aptamer is freed from an anchor aptamer. Nanofiltration membranes can provide is nM pore sizes, and ultrafiltration 10s to 100s nm pore sizes (PES, track-etch, and other materials), resulting in size selective permeability that would enable mainly only the signaling aptamer 882 to penetrate the hydrogel or membrane 838.

And so, still referring to FIG. 8 , when analyte 19 is not bound to the anchor aptamer 884, the signaling aptamer 882 remains associated with the anchor aptamer 884, and so the redox tag 840 on signaling aptamer 882 is less available (or not available) for electron transfer with the electrode 850 (because the signaling aptamer 882 will be unable to cross membrane 838 due to being complexed with anchor aptamer 884). However, once the anchor aptamer 884 binds to analyte 19, the signaling aptamer 882 is released from anchor aptamer 884 and is able to pass through membrane 838, resulting in the redox tag 840 becoming available for electron transfer with the electrode 850. Further, while the embodiment shown in FIG. 8 depicts analyte 19 binding to anchor aptamer 884 and redox tag 840 on signaling aptamer 882, in an alternate embodiment analyte binding may occur with signaling aptamer (signaling aptamer having redox tag), and binding of analyte to signaling aptamer may serve to release signaling aptamer from anchor aptamer (such as by change in conformation of signaling aptamer).

With reference to FIG. 9 , where like numerals refer to like features, another embodiment in accordance with principles of the present invention is shown. In certain of the various embodiments discussed herein, a membrane is used to selectively allow passage of certain molecules and not of others. However, as no membrane is perfectly size selective, and as aptamers and redox tags can degrade over time, it may be advantageous to continually introduce a fresh supply of aptamers, signaling aptamers, and/or anchor aptamers or other solutes that increase performance of the sensor or improve longevity of the sensor (e.g., nuclease inhibitors, for example). Thus, as shown in FIG. 9 , a portion of a device 900 includes substrates 910, at least one electrode 950, a membrane 936, a sample fluid 14, a sensor fluid 18, and a reservoir fluid 17. The membrane 936 exhibits mass flow represented at reference numeral 991, and the device also includes a diffusion restrictive feature 935 (such as a pinhole or membrane) with a mass flow represented at reference numeral 993.

As a nonlimiting example of that shown in FIG. 9 , consider a 0.2 kDa dialysis membrane for membrane 936 and assume the aptamers are 10-10× larger than the solute to be detected (e.g., phenylalanine, cortisol, etc.). Assume the system is designed such that the volume of reservoir fluid 17 is at least one of 2×, 10×, 50×, or 250× greater than volume of sensor fluid 18 and that the mass flow 991 of aptamer is at least 2×, 10×, 50×, or 250× less than mass flow of aptamer 993, while the mass flow 991 of the analyte is at least 2×, 10×, 50×, or 250× greater than the mass flow of the analyte 993. As a result, the concentrations of analyte will be within at least 50%, 10%, 2%, or 0.4% of each other when comparing sample fluid 14 with sensor fluid 18, and the concentrations of aptamer will be within at least 50%, 10%, 2%, or 0.4% of each other when comparing sensor fluid 18 and reservoir fluid 17.

As a geometrical example, consider a membrane 936 with 0.2 cm² area and 10% porosity to the analyte, and a diffusion restrictive feature 935 that is a pinhole in materials 910 and 950 0.001 cm² in area and 0.001 cm in length. The mass transport for a small analyte through the membrane will be equivalent to 0.02 cm² area and the mass transport through the feature 935 0.001 cm², which is 20× different, satisfying the above stated criteria for design as shown in FIG. 9 . As a result, both analyte and aptamer concentrations can be maintained for prolonged periods of times (days, weeks, months) even if aptamer is lost from the device or degraded over time. Aptamers could also degrade over time and their presence in the device and the presence of other contaminants such as nucleases or proteins could be problematic. For example, if signaling aptamers became cleaved and their molecular weight decreased, they could give a false higher reading of signal in embodiments of the present invention. With membrane protection of the sensor fluid from the sample fluid, most degradation or contamination modes will be very slow, such that the reservoir may also act as a waste removal element.

The various embodiments disclosed herein can be enabled to be user-calibrated, factory-calibrated, or calibration-free. User-calibration could for example require a pin-prick blood draw and running of a conventional assay to measure analyte concentration, and that concentration data entered into the software that runs the sensing device.

Factory-calibrated implies that the device requires calibration, but that the calibration is shelf-stable and stable for at least a portion of the use period of the device. Embodiments, such as those shown in FIG. 5B and FIG. 5C, could benefit from factory calibration if they are interrogated by square wave voltammetry, and if the passivation layer 548 thickness is kept fairly constant (e.g., using a mercaptohexanol passivation layer 548 or polyethylene glycol terminated passivation layer 548 that is resistant to fouling). In factory calibration, the device is tested with a sample fluid with a known concentration of analyte, and that information is then shipped along with the product in order to enable it to start its use with proper calibration.

Calibration-free operation is possible if one could eliminate the factors that could cause a sensor signal to drift or change. Considering the embodiments of FIGS. 6 and 9 , the aptamer concentrations can be kept constant, and with a chronoamperometric measurement response the change in current vs. time will be dependent on diffusion coefficient of the signaling aptamer 672 vs. the anchor aptamer 674 and signaling aptamer 672 bound together. The diffusion coefficient will not change in a sample fluid such as interstitial fluid, and an overvoltage can be supplied to measure the chronoamperometric response even if thickness of the passivating layer 648 changes slightly. Simply, the chronoamperometric response will measure the percentage of signaling aptamer 672 that is free from the anchor aptamer 674, which is directly related to the binding affinity of the analyte to the aptamers, hence enabling calibration-free operation because the concentrations of the signaling aptamers 672 and anchor aptamers 674 are known. Calibration-free operation is also possible using the constructs of FIG. 5D or FIG. 5E by measuring changes in electron transfer rates, peak position shifts, or ratios (not individual magnitudes) of two or more redox peaks from different redox tags 546, 548 (FIG. 5E).

As described above, certain aspects of the present invention are directed to devices that include a feature of continuous solute passivation. As an example, in an electrochemical aptamer sensor, background current can increase by 3× or more in just 2 hours in a buffer solution. However, when an embodiment of the present invention with continuous solute passivation is used in serum, background current may only increase by 1.3× or not at all. Solutes in various sample fluids (e.g., interstitial fluid, blood, or others), such as peptides, amino-acids, and albumin, (i.e., endogenous passivation) can continuously passivate an electrode of an electrochemical aptamer device for a period of time that is greater than the duration of passivation provided by an exogenous passivating layer alone such as mercaptohexanol. This may serve to extend the lifetime of such a device.

However, as described previously, serum or biofluids may also contain proteases, enzymes, and other solutes that can degrade a sensing device over time by attacking the aptamers, or by creating passivating or fouling layers that are too thick to allow operation of the device (e.g., due to too much protein passivation or fouling of the electrode). As illustrated in FIG. 3 , a membrane 336 can allow continuous solute passivation by being an ultra-filtration, nanofiltration, dialysis, oil-impregnated membrane, or other type of membrane that would pass an analyte of interest such as cortisol or phenyalanine, while performing one or more additional functions such as: (1) passing only solutes from the sample fluid 14 that in some cases enable an ideal passivation of the electrode (e.g. small molecules instead of proteins); and (2) retaining at least one solute in the sensor fluid 18 near the electrode 350 such that they can continuously passivate the electrode 350 as needed. In an embodiment, the membrane is configured to retain at least one solute in the sensor fluid 18 within at least 500 μm of the electrode. Simply, the membrane 336 could have a molecular weight cutoff that is near or greater than the analyte and the passivating solutes, or alternately if the passivating solutes are to be held in the sensor fluid the membrane 336 could have a molecular weight cutoff that is near or greater than the analyte and lesser than the passivating solutes. For example, the membrane 336 could have a molecular weight cutoff of 1 kDa, which would block proteases (15-30 kDa) from the interstitial fluid in the dermis 12 b, while trapping in albumin which is able to continuously passivate the electrode 350. Alternately, the reservoir fluid 17 of FIG. 9 could continuously introduce albumin, peptides or non-natural chemical solutes with single or multiple thiol-bonding sites to a gold or platinum electrode 350, or other suitable solutes to continuously passivate the electrode 350, 950. Simply, the membrane 336 could have a molecular weight cutoff that less than at least one solute for passivation that is introduced from the reservoir fluid 17 to the sensor fluid 18, the at least one solute continuously passivating the electrode 350.

In embodiments not including such a membrane (or in embodiments including membrane—but ones which may still result in the passage of solutes that can ultimately foul the device over time), any passivation layer may continue to grow in thickness over time, which at some point will decrease electron transfer from the redox tags. As described above, this not only can reduce signal strength from the sensor, but can also result in errors in reading of the concentration of analytes. At some point, surface fouling of one or more of the electrodes could become a bottleneck. Further, techniques used to prevent such issues enzymatic sensors simply may not apply to solution-phase aptamer sensors, because an enzyme can be ‘buried’ in a protective material such as a hydrogel and still retain its enzymatic activity, whereas an aptamer cannot be fully buried in a material because its signal transduction depends on its freedom of movement in solution. And so, an aspect of the present invention provides the ability to clean one or more electrodes in-situ of naturally passivating electrode foulants, (i.e., during use of the device). In various embodiments directed to this aspect of the present invention, working, counter, reference, or other electrodes (not all shown in the figures) may be electrically or electrochemically cleaned in-situ with connection to an electronic source (not shown). Voltages can be applied such as 1V, 2V, or other suitable voltages to briefly cause electrolysis, for example with durations of microseconds to seconds, in order to clean the electrode surface while not damaging the device 500. For example, a device with an electrode having a surface area 10× smaller than a surface area of the membrane. Alternatively, the device may include a membrane with a membrane area of 10 mm² and a smaller electrode area of 100 μm² could clean the tiny electrode without affecting much of the sample fluid across the rest of the device (e.g., pH effects, gas bubbles, etc.). As further example, a diamond electrode could be cleaned with dozens of current pulses, for example current pulses of 10 mA/cm², for 100 ms with a duty cycle of 80-95% for the pulses. Typically, corrosion-resistant (noble) metals are used in biosensing such as carbon-based, diamond, gold, or platinum with a volts vs. saturated calomel electrode that are >0 V.

In an alternative embodiment, titanium, nickel, steel, copper, silicon bronze, or other metals or alloys with <0 V volts vs. saturated calomel electrode can be used such that their natural corrosion at blood pH levels will gradually remove (clean) an electrode fouling layer over time with or without electrochemical cleaning methods. These corrosion processes can be electrochemically increased or decreased by use of low pulsed or DC voltages, e.g., via anodization vs. etching. In yet another alternate example embodiment, an electrode can be used such as gold or platinum on which electrochemically and oxide layer is formed and then removed. Such a process could involve, for example, a positive potential to form a monolayer or more of oxide, an anodic potential and then a cathodic potential, which can be performed once or a plurality of times in series. Assume that each time the electrode is cleaned 0.3 to 3 nm of metal needs to be removed, then the thickness of the electrode may place a limit on how many times the electrode can be cleaned by repeat oxidative cleaning. If an electrode were 0.3 to 30 μm thick then it could be cleaned 100 to 100,000 times. The electrode cleaning could be required at every data point measurement (e.g., every 2 minutes), at least once every 24 hours, (which may be only once every 24 hours) depending on the application, which means the electrode itself could have an operational life that is at least 200 minutes (more than one hour) or even greater than 1 week or greater than 6 months.

Turning now to FIGS. 10A and 10B, embodiments where working, counter, reference, or other electrodes may be mechanically cleaned in-situ are shown. With reference first to FIG. 10A, a device 1000 a includes a substrate 1010 having an electrode 1050 adjacent thereto. Device 1000 a also includes a membrane 1036 between sensor fluid 18 and sample fluid 14. A cleaning element may include a ceramic or gold disk 1090 and an associated hydrogel or brush 1092 that can mechanically clean the surface of electrode 1050. In an embodiment using a brush 1092—the brush may be a PET polymer brush. Mechanical cleaning requires motion, and the brush 1092 may be configured to move in response to a stimulus which can be applied via conventional means (e.g., a motor, thermally responsive polymer, etc), or more elegantly for an implanted sensor device 1000 using external stimuli.

Turning now to FIG. 10B, an alternate embodiment is shown. In FIG. 10B, a device 1000 b includes a substrate 1010 having an electrode 1050 adjacent thereto. Device 1000 b also includes a membrane 1036 between sensor fluid 18 and sample fluid 14. A cleaning element may include small, micro- or nano-beads 1094 that mechanically clean the surface of electrode 1050. Mechanical cleaning requires motion, and the microbeads or nanobeads may be configured to move in response to a stimulus which can be applied via conventional means (e.g., a motor, thermally responsive polymer, etc), or more elegantly for an implanted sensor device 1000 using external stimuli. For example, mechanical cleaning elements 1090, 1094, could have a density, in different embodiments, that is at least >10%, >100%, or >1000% different than the density of water (e.g., gold, or air-filled ceramic beads, etc.), such that a stimulating element, such as an external mechanically vibrating element, or sonic element, can vibrate elements 1090, 1094, against electrode 1050. Even more elegant, is allowing natural body motion or position change to cause elements 1090, 1094 to move and clean electrode 1050. Elements 1090, 1094 may be magnetic or electromagnetic in property (e.g. magnetic bead materials) and moved via magnetic field from an external electromagnetic transducer.

In some embodiments, mechanical cleaning is only used as needed (daily, weekly, monthly, etc.). In another embodiment, such as, e.g., for an indwelling needle sensor (partially implanted, like Abbott's Freestyle Libra wearable), at least one electrode, such as a wire electrode, has a rubber or brush sheath, and the wire electrode mechanically extends or retracts through the sheath to clean the electrode as needed. In this embodiment, the mechanical transduction may be provided by an external solenoid located in the wearable device.

In certain embodiments of the present invention, an electrode may be inoperable for measurement of analyte while it is being cleaned. And so, in some embodiments, the present invention may include a plurality working electrodes (not shown in FIGS. 10A and 10B) each being cleaned and used for sensor measurement at independent times. As a non-limiting example, an electrode may be cleaned, then after at least 1 hour of physi-absorbtion of sensor fluid solutes onto the surface, used as a sensing electrode for 1 day before being cleaned again. With this example, only two electrodes would need to be interchangeably cleaned.

With respect to embodiments of the present invention, knowing when to clean an electrode is also of value. In some embodiments of the present invention, electrical impedance of the electrode may be continuously measured to determine when a fouling layer is too thick. In other embodiments, electrical impedance may also be used to correct for sensor drift as fouling layer thickness increases. As a non-limiting example, an additional redox couple with a different redox potential than the redox couple for the aptamer (such as aptamer and redox couple 370, 372 of FIG. 3 ) is used with an aptamer that does not respond to changes in analyte concentration, or just floating in solution or attached to some other solute or particle, which is used to indirectly measure fouling layer thickness to determine need for cleaning or sensor drift correction. Measurements such as electron transfer rates, shifts in redox peak potentials, or other methods may be used that provide a measure of fouling or passivating layer thickness.

With respect to embodiments of the present invention, the membranes that isolate the sample fluid from the sensor fluid can also be subject to fouling. This can, in turn, increase device lag time or even skew analyte concentrations if cells form on the membrane. Therefore, the methods taught for cleaning the electrodes in the present invention could also be applied to cleaning the surface of any membrane present in the device (such as any embodiments of devices and membranes taught herein). For example, the membrane could be a material similar to a dialysis membrane with a molecular weight cutoff of 300 Da. Onto this membrane a nano-colloidal ink such as silver or another metal can be printed and sintered at low temperatures or near room temperature (as is enabled by nano-colloidal inks), but not fully sintered such that resulting metal film remains nanoporous. This creates an electrode on the membrane that can be cleaned using one of the methods as taught previously. The electrode itself could be the membrane as well with properly tuned porosity. Other electrode coated membranes such at Pt coated Nafion membranes are possible as well. Therefore, generally the present invention may include in-situ cleaning of one electrode that is also a membrane, or which is coated onto a membrane. In an embodiment, the cleaning element includes a membrane housing the electrode, or, alternatively or in addition, the cleaning element is coated onto a membrane housing the electrode.

EXAMPLES Example 1

With reference to FIGS. 11A and 11B a cortisol binding aptamer was utilized in a manner similar to that taught in FIG. 6C, where the signaling aptamer 672 was tagged with methylene blue as a redox tag 640 with an aptamer sequence of GTCGTCCCGAGAG [SEQ ID NO.1] and where the anchor aptamer 674 with a sequence of ctctcgggacgacGCCCGCATGTTCCATGGATAGTCTTGACTAgtcgtccc [SEQ ID NO. 2]. Electrodes 650, 652 were gold interdigitated electrodes with a 5 μm spacing in between them. The gold electrodes were passivated with an exogenous molecule of mercaptohexanol. No hydrogel 638 was utilized in this experiment. The sensor solution was buffer solution with 5 μM of the aptamers 650, 652 in solution, and a reference electrode of platinum was used. The device 600 was measured amperometrically vs. a titration curve of cortisol as the analyte 19. The results are shown in FIG. 10A and FIG. 10B (open circles, open diamonds, and solid diamonds), and a control experiment with titration of simply adding more cortisol but without aptamer in solution is also shown in FIG. 10B (solid circles). The signal gain in Example 1 is as much as 70%, and if the anchor aptamer was made even larger or smaller the signal gain could be tuned to be as much as 200% or more as little as 5% based on the change in diffusion rate of the signaling aptamer to the electrode compared to the signaling aptamer when it is bound to the anchor aptamer. Signal gain is also measured above a baseline signal, and changing signaling aptamer concentration can therefore be used to tune the signal gain.

Example 2

The experiment of Example 1 was repeated but instead of using mercaptohexanol passivation of the gold electrodes 650, 652, endogenous small molecule solutes found in blood or interstitial fluid were allowed to passivate the gold electrode 650, 652. It was found that without passivation background current was very high, but that both mercaptohexanol and endogeneous solutes were able to adequately reduce background current and enable sensor operation.

Although not described in detail herein, other steps which are readily interpreted from or incorporated along with the disclosed embodiments shall be included as part of the invention. The embodiments that have been described herein provide specific examples to portray inventive elements, but will not necessarily cover all possible embodiments commonly known to those skilled in the art. 

What is claimed is:
 1. A device for detecting the presence of, or measuring the concentration of, at least one analyte in a sample fluid, the device comprising: at least one electrode; a sensor fluid in communication with the at least one electrode, the sensor fluid including a plurality of aptamers freely diffusing in the sensor fluid; and at least one element configured to passivate the at least one electrode by continuous solute passivation.
 2. The device of claim 1, wherein the element includes a membrane that is impermeable to at least one passivating solute adjacent to the at least one electrode.
 3. The device of claim 1, wherein the element includes a membrane that is permeable to at least one passivating solute adjacent to the at least one electrode.
 4. The device of claim 1, wherein the element includes one or more solutes of a plurality of solutes endogenous to a sample fluid.
 5. The device of claim 4, wherein the sample fluid is one of blood or interstitial fluid.
 6. The device of claim 2, wherein the membrane has a molecular weight cutoff that is less than the at least one element.
 7. The device of claim 3, wherein the membrane has a molecular weight cutoff that is greater than the at least one element.
 8. The device of claim 1, further comprising at least one reservoir fluid.
 9. The device of claim 8, wherein the reservoir fluid contains the at least one element.
 10. The device of claim 1, wherein one or more aptamers of the plurality of aptamers each include a redox tag; and wherein the device further includes at least one membrane in communication with the sensor fluid and adapted to be in communication with a sample fluid, wherein the membrane is permeable to analyte in a sample fluid, and is impermeable to each aptamer of the plurality of aptamers.
 11. The device of claim 1, wherein the plurality of aptamers comprise a plurality of signaling aptamers and a plurality of anchor aptamers.
 12. The device of claim 11, further comprising a plurality of redox tags, wherein each redox tag of the plurality of redox tags is bound to a signaling aptamer of the plurality of signaling aptamers.
 13. The device of claim 10, wherein the membrane has a molecular weight cutoff of <1000 Da.
 14. The device claim 1, further comprising a housing having one or more interior chambers containing the at least one electrode, sensor fluid, and element configured to passivate the at least one electrode; and wherein the housing is adapted to be placed outside of the body and the stratum-corneum of the skin of a subject.
 15. The device of claim 1, further comprising a housing having one or more interior chambers containing the at least one electrode, sensor fluid, and element configured to passivate the at least one electrode; and wherein the housing is at least a portion of an in-dwelling device.
 16. The device of claim 15, further comprising one or more microneedles in fluid communication with the one or more interior chambers.
 17. The device of claim 1 further comprising a housing having one or more interior chambers containing the at least one electrode, sensor fluid, and element configured to passivate the at least one electrode; and wherein the housing is adapted to be implanted into a subject.
 18. The device of claim 1, wherein the at least one element configured to passivate the electrode is located in a passivating layer adjacent to the electrode.
 19. The device of claim 1, wherein the at least one element comprises an exogenous molecule.
 20. The device of claim 1, wherein the at least one element comprises an endogenous solute from the sample fluid.
 21. The device of claim 20, wherein the endogenous solute is configured to leave the electrode and be replaced by another molecule.
 22. The device of claim 10, wherein the membrane has a molecular weight cutoff of chosen from at least one of less than 300 Da, less than 1000 Da, less than 3 kDa, less than 10 kDa, less than 30 kDa, less than 100 kDa, and less than 300 kDa.
 23. The device of claim 10, wherein the membrane is configured to allow only solutes from the sample fluid to permeate therethrough, and wherein the solutes from the sample fluid include the analyte.
 24. The device of claim 10, wherein the membrane is permeable to the analyte and wherein the membrane is positioned to retain the at least one element within at least 500 μm of the electrode.
 25. The device of claim 1, further comprising a reservoir fluid in fluid communication with the sensor fluid.
 26. The device of claim 25, wherein the reservoir fluid includes albumin, peptides, or non-natural chemical solutes with single or multiple thiol binding sites, and is configured to continuously introduce the albumin, peptides, or non-natural chemical solutes to the electrode.
 27. The device of claim 26, wherein the reservoir fluid is configured to supply the at least one element to the sensor fluid, and wherein the membrane has a molecular weight cutoff less than the at least one element.
 28. The device of claim 26, wherein the reservoir fluid is configured to accept waste from the sensor fluid.
 29. The device of claim 1, wherein the plurality of aptamers further comprising one or more anchor aptamers immobilized via a linkage to a hydrogel.
 30. The device of claim 10, wherein the membrane has a selective permeability based on size, charge, or at least one other property of the at least one element.
 31. A device for detecting the presence of, or measuring the concentration of, at least one analyte in a sample fluid, the device comprising: a plurality of aptamers disposed in a sensor fluid; a plurality of redox tags, wherein each redox tag of the plurality of redox tags is individually coupled to an aptamer of the plurality of aptamers, each redox tag configured to change an electron transfer to between the electrode and the aptamer in response to the analyte coupling to the aptamer; at least one electrode in communication with the sensor fluid; and at least one cleaning element configured to clean a surface of the electrode.
 32. The device of claim 31, wherein the at least one electrode further comprises a working electrode, a counter electrode, or a reference electrode.
 33. The device of claim 31, further comprising a membrane at least partially housing the electrode, the electrode having a surface area 10× smaller than a surface area of the membrane.
 34. The device of claim 31, wherein the electrode comprises titanium, nickel, or steel, copper, silicon bronze, or other metal or alloy with <0 V volts vs. saturated calomel, and wherein the electrode is configured to remove an electrode fouling layer from the electrode.
 35. The device of claim 31, wherein the electrode is 0.3 to 30 μm thick.
 36. The device of claim 31, wherein the cleaning element is configured to clean the electrode at least one of at least once every 24 hours.
 37. The device of claim 31, wherein the electrode is configured to have an operational life that is at least 200 minutes, greater than 1 week, or greater than 6 months.
 38. The device of claim 31, wherein the cleaning element is a polymer brush configured to mechanically clean the surface of the electrode.
 39. The device of claim 38, wherein the polymer brush comprises microbeads or nanobeads configured to mechanically clean the surface of the electrode.
 40. The device of claim 39, wherein the microbeads or nanobeads are configured to move in response to a stimulus from a motor, thermally responsive polymer, or external stimuli.
 41. The device of claim 31, wherein the cleaning element has a density that is at least greater than 10%, greater than 100%, or greater than 1000% different than the density of water.
 42. The device of claim 31, further comprising a stimulating element configured to mechanically vibrate or sonically vibrate the cleaning element.
 43. The device of claim 31, wherein the cleaning element is configured to clean the electrode in response to a natural body motion or position change of a user of the device.
 44. The device of claim 31, wherein the cleaning element is magnetic or electromagnetic and is configured to be moved in response to a magnetic field from an external electromagnetic transducer.
 45. The device of claim 31, wherein the at least one electrode comprises a plurality of electrodes, each electrode of the plurality of electrodes configured to be cleaned at independent times and configured to be used for sensor measurement at independent times.
 46. The device of claim 45, wherein at least one of the plurality of electrodes is configured to be used as a sensor after at least 1 hour of physi-absorption of the sensor fluid onto the surface of the electrode following cleaning of the electrode.
 47. The device of claim 46, wherein the electrode is configured to be used as a sensing electrode for at least 1 day prior to a subsequent cleaning.
 48. The device of claim 31, wherein the device is configured to continuously measure an electrical impedance of the electrode.
 49. The device of claim 48, further comprising a second plurality of aptamers, each aptamer of the second plurality of aptamers configured to not respond to changes in analyte concentration in the sample fluid, and each aptamer of the second plurality of aptamers comprising a second redox tag having a redox potential different from the redox tag coupled to the aptamers of the plurality of aptamers, the second plurality of aptamers configured to measure a fouling layer thickness on the surface of the electrode.
 50. The device of claim 31, wherein the cleaning element is located in the sensor fluid, in-situ.
 51. The device of claim 31, wherein the cleaning element comprises a membrane housing the electrode or is coated onto a membrane housing the electrode.
 52. A method of sensing an analyte in a sample fluid, the method comprising: bringing an analyte in the sample fluid into contact with a plurality of aptamers disposed in a sensor fluid, the contact of the plurality of aptamers with any analyte resulting in a change in electron transfer between a redox tag attached to an aptamer bound to analyte and an electrode; and measuring the change in electron transfer between the redox tag and the electrode; wherein the electrode is passivated by continuous solute passivation.
 53. The method of claim 52, wherein the measuring of the change in electron transfer occurs by voltammetry, scanning wave voltammetry, amperometry, chronoamperometry, coulometry, chronocoulometry, or combinations thereof.
 54. The method of claim 53, further comprising cleaning a working electrode, a counter electrode, or a reference electrode in-situ.
 55. The method of claim 54, wherein the cleaning is performed electrically or electrochemically in connection to an electronic source.
 56. The method of claim 54, wherein the cleaning includes removing 0.3 to 3 nm from the electrode.
 57. The method of claim 54, wherein the cleaning includes pulsing a current. 